Probiotics for use in expecting female mammals for enhancing the immunity of their offspring

ABSTRACT

The present invention relates to the use of probiotic on expecting female mammals to boost the immune status of an offspring. The use can induce an enhanced response of the offspring after birth to an infectious antigenic exposure. Ultimately the use of probiotic in expecting females can induce a better protection of their offspring against infectious diseases.

PRIORITY CLAIM

The present application is a divisional application of U.S. patentapplication Ser. No. 12/935,149, filed on Sep. 28, 2010, which is aNational Stage of International Application No. PCT/EP09/52869, filed onMar. 11, 2009, which claims priority to European Patent Application No.08153566.8, filed Mar. 28, 2008, the entire contents of each of whichare being incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the use of probiotics bacteria in themanufacture of a nutritional composition for expecting female mammals toenhance the immunological status in their newborn offspring.

BACKGROUND

The immunological status of newborn is an important issue. This statusencompasses to the protection present at the birth of the infant and tothe acquisition of such immunological protection during the first hours,days or weeks of the infant life. The ability to acquire and maintainsuch protection is a crucial factor in the health of the infant facedwith his new environment. In humans these issues are of the highestimportance for the health of the population.

Maintaining the expecting mothers in good health condition duringgestation is a key factor for promoting the health of the offspringduring gestation and after birth. General health factors that are knowninclude their nutritional habits, their micro-nutriment intake, theirinfectious history and also relate to their immunological status. Forexample deficiencies in some minerals, vitamins or substances (such asfolic acid) can affect the development of the fetus and also affectpost-natal infant development. The nutrition of the mother plays in thatrespect a key role in the future health of the infants and much effortshave been made to monitor and improve the nutritional balance of theexpecting mothers. Food supplements or simply general eating guidelinesare essential in that respect.

Along with general nutritional guidance for expecting mothers, it is nowrecognized that some specific food can promote the proliferation of aspecific microbiota in the gastro-intestinal tract of the expectingmother. The balanced microbiota in turn may have an effect on the host.

The use of prebiotics, i.e. nutritional substances for improving theintestinal microbiota of a host, has been described for example in WO02/07533 for its health benefits in females.

Moreover, it has been claimed in WO2007/105945A that feeding of theexpecting mothers with prebiotics ingredients, especially certain typeof non digestible saccharides could improve the microbiota and/or theimmune system of the offspring.

Complementing the nutritional intake with micro-organisms, preferablylive micro-organisms, has also been demonstrated to improve themicrobiota balance of the intestinal tract of the host. The modulationof the microbiota of the intestinal tract by specific livemicro-organisms has been shown to bring particular positivephysiological effects. For example the immune response induced byspecific types of probiotic bacteria has been widely studied anddescribed (“Cross talk between probiotics bacteria and the host immunesystem”, The journal of nutrition. Blaise Corthesy et al., supplement,2007, pages 781S-790S). As described in the scientific literature,specific strains of micro-organisms have been shown more particularly tohave beneficial effects. Bifidobacterium, Lactobacillus acidophilus, andLactobacillus caseii are generally known examples of families shown tohave probiotics effects.

It is hypothesized that probiotics, as other commensals, will influenceimmune functions of the host either via modulation of the microbiotacomposition and/or metabolic activity or through a direct interactionwith the immune system underlying the gut mucosa. Following thisinteraction, immune functions will be activated as reflected by therelease of immune mediators (cytokines), production of antibodies andactivation of lymphocytes as well as other immune cells. These activatedcells, cytokines and/or bacterial compounds will exert immune modulatoryfunctions at different location of the body through the bloodcirculation. In that respect, it is postulated that a beneficial effecton the immune status of the mother through probiotic supplementation mayalready influence fetal immune development. Moreover, it is also knownthat immune cells and other bioactive factors originating from the gutof the mother could be transported to the breast milk via anentero-mammary transport pathway and transmitted to the neonate duringlactation. Therefore, it is worth to further hypothesize thatsupplementation of pregnant mothers might promote an enrichment of thebreast milk with immune factors that would contribute to supportneonatal immune development.

Securing the healthy future of offspring is a well recognized need. Morespecifically insuring the best development and maturation of the immunesystem of the offspring is of the highest importance.

Conventionally, beside medical treatments responding to specific medicalconditions, emphasis is put on a good nutritional balance of theexpecting mothers. Not much is however known on how to specificallyenhance the immune status of the offspring during the gestation period.

There is however a need for a further step in securing the healthyfuture of the offspring, using the most recent finding on nutrition.

There is therefore a need for positively impacting the health of theoffspring by a targeted nutritional diet of the expecting mothers.

There is in particular a need to help guaranteeing the best immunesystem in the offspring, in order to best prepare them to the early lifeantigenic challenges as well as to enhance the future maturation oftheir immune system to better promote protection during later infancy.

There is a need to impact the building of the immune system of theoffspring at the earliest possible stage during and all along thegestation as well as the early phases of their new born life when theimmune system is maturing at high pace.

There is a need to boost the ability of the immune system of theoffspring to react against antigens in general and against infectiousdiseases in particular.

There is a need to deliver these benefits by means that are bothefficient and deprived of negative impact on the expecting mothers andor their offspring.

SUMMARY

In a first aspect, the present invention provides the use of probioticson expecting female mammals in the manufacturing of a composition forboosting the immunity of their offspring after birth.

In a second aspect, the present invention provides the use of specificprobiotics organisms such as Bifidobacterium, Lactobacillus,Streptococcus, Enterococcus and Saccharomyces, in expecting femalehumans for boosting the immunity of their offspring after birth.

In a third aspect, the present invention provides the use of theprobiotics during the gestation period and/or during the lactationperiod of the offspring.

In another aspect of the invention the probiotics are administered in orwith the food or drinks of the expecting mother, and together with otheractive ingredients such as prebiotics or other dietary supplements.

The invention further extends to the composition used by the abovemethods for the purposed cited.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 3 highlight schematically the experimental procedures of thestudies 1 and 2 respectively described.

FIGS. 2 and 4 show the results of the studies indicating an immune boostin the offspring. In FIG. 4, A shows the systemic immune responses ofnon-antigen specific spleen T cell at 3 weeks of proliferation, and Bshows the anti-TT lgG antibody responses at 4 weeks after the firstimmunization (left panel) and 2 weeks after the boost (right panel).

DETAILED DESCRIPTION

Definitions:

In this specification, the following terms have the following meanings:

“Expecting female mammals” are female mammals that have at least onefecunded ovocyte in development in their uterus. Preferentially, femalehumans (i.e. mothers or mothers-to-be) are considered by this invention.

“Probiotic” means microbial cell preparations or components of microbialcells with a beneficial effect on the health or well-being of the host,(Salminen S, Ouwehand A. Benno Y. et al “Probiotics: how should they bedefined” Trends Food Sci. Technol. 1999:10 107-10). The probiotic cancomprise a unique strain of micro-organism, of a mix of various strainsand/or a mix of various micro-organisms. In case of mixtures thesingular term “probiotic” can still be used to designate the probioticmixture or preparation.

“Prebiotic” generally means a non digestible food ingredient thatbeneficially affects the host by selectively stimulating the growthand/or activity of micro-organisms present in the gut of the host, andthus attempt to improve host health.

“Offspring” relates to the newly-born or to-be-born infant from thesubject female mammals. In particular, it includes the progeny still ingestation. Preferentially the juvenile phase/infancy phase is consideredby the present invention (i.e. up to adolescence in humans—12-14 years),more preferentially the present invention relates to the immune statusof the offspring in the early infancy (up to 2 to 4 years of age inhumans).

The inventors have found that the administration of probiotics toexpecting female mammals can impact the immune system of the offspring,and more particularly can boost the immune system of the offspring suchas to enable a better and stronger response of the immune system afterexposure to antigens. The modulation of immune system of the offspringcan happen very early in their intrauterine life or early phase of extrauterine life, when their immune system is maturing.

The inventors have evidenced the boosting effect in absence of obviousdirect contact between the intestinal system of the offspring and theprobiotics composition administered to the expecting females, i.e.during the intra-uterine life of the offspring.

Further the inventors have found that such composition comprisingprobiotics can also have a beneficial effect on the immune system of theoffspring during the lactation period.

Without being bound by the theory, it is speculated that the partialcolonization of the intestinal tract of the expecting females by theprobiotic induces the creation of a molecular signal. It is believe thatthe offspring receives and reacts to this molecular signal. It isfurther believed that the immune system of the offspring is affected bythat signal. That signal has a positive effect on the maturation of theimmune system of the offspring. This can lead to a better ability torespond to antigens after birth. It is further speculated that thesignal can be transmitted to the offspring during the lactation period,more specifically if there was a preconditioning to the signal duringthe gestation period of the offspring.

Expecting Females.

The use of and the composition of the present invention is performed onand administered to expecting females mammals, i.e. females which aredue to give birth to offspring. The use considered by the presentinvention extends from the conception of the offspring (fecundation)through the full gestation period up to the delivery of the offspring.It can also extend to the lactation period up to the weaning. Partialweaning, up to the full termination of the lactation, is also includedin the present invention. It is not excluded that the use considered bythe invention also extends to the period immediately preceding thefecundation that has an impact on the health status of the females, andindirectly on the health status of the offspring.

The mammals can be pregnant humans females. In such case the gestationperiod is about 9 months and the lactation period can considerably varyaccording to habits, culture and health status of the females. Thefemale mammals can also be other female mammals including horses andpets such as cats and dogs. Other expecting female mammals are notexcluded from this invention.

Probiotic Micro-Organisms.

The probiotic micro-organisms considered by this invention can includeany probiotic selected form the group comprising of Bifidobacterium,Lactobacillus, Streptococcus, Enterococcus and Saccharomyces or mixturesthereof, preferably selected from the group consisting ofBifidobacterium longum, Bifidobacierium lactis, Lactobacillusacidophilus, Lactobacillus rhamnosus, Lactobacillus paracasei,Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillussalivarius, Lactobacillus reuteri, Enterococcus faecium, Streptococcussp. and Saccharomyces boulardii or mixtures thereof. Other probioticmicro-organisms are not excluded from this invention provided they areable to deliver the immune boost effect described.

More preferably the probiotic is selected from the group comprising ofLactobacillus rhamnosus CGMCC 1.3724 (nick name NCC4007 and LPR),Bifidobacterium lactis CNCM 1-3446 sold inter alia by the ChristianHansen company of Denmark under the trade mark Bb12 (nick mane NCC2818),Bifidobacterium longum ATCC BAA-999 sold by Morinaga Milk Industry Co.Ltd. of Japan under the trade mark BB536. Lactobacillus johnsonii CNCM1-1225 (nick name NCC533 and La1), Lactobacillus fermentum VRI 003 soldby Probiomics (Australia), under the trademark PCC, Bifidobacteriumlongum CNCM 1-2170, Bifidobacterium longum CNCM 1-2618, Bifidobacteriumbreve sold by Danisco (Denmark) under the trade mark Bb-03,Bifidobacterium breve sold by Morinaga (Japan) under the trade markM-16V and the strain of Bifidobacterium breve sold by Institut Rosell(Lallemand) (Canada) under the trade mark R0070. Lactobacillus paracaseiCNCM 1-1292. Lactobacillus rhamnosus ATCC 53103 obtainable inter aliafrom Valio Oy of Finland under the trade mark LGG, Enterococcus faeciumSF 68, and mixtures thereof. A preferred probiotic is Lactobacillusrhamnosus CGMCC 1.3724.

Doses of Probiotic.

The probiotic can be present in the composition in a wide range of %provided that the specific probiotic used deliver the immunity boostingeffect described. However, preferably, the probiotic is present in thecomposition in an amount equivalent to between 10³ and 10¹⁰ cfu/g of drycomposition (cfu=colony forming unit). This expression includes thepossibilities that the bacteria are live, inactivated or dead or evenpresent as fragments such as DNA or cell wall materials. In other words,the quantity of bacteria which the formula contains is expressed interms of the colony forming ability of that quantity of bacteria as ifall the bacteria were live irrespective of whether they are, in fact,live, inactivated or dead, fragmented or a mixture of any or all ofthese states. Preferably the probiotic is present in an amountequivalent to between 10⁴ to 10⁹ cfu/g of dry composition, even morepreferably in an amount equivalent to between 10⁶ and 10⁸ cfu/g of drycomposition.

Method of Administration.

The composition can be administered to the expecting females by avarious ways as long as it induces a contact between the composition andthe gastro-intestinal tract of the females. Preferably the compositionis administered as part of the food, drinks or dietary supplements ofthe females. The composition can also be administered in apharmaceutical composition. Preferably the administration is oral orenteral. Oral administration is most preferred as it has a lesstraumatic impact on the females. However in pathological conditions orwhen enteral feeding is otherwise used, the administration of thecomposition can be added to the enteral feeding.

Administration with Other Compounds.

In any case, the composition can be administered alone (pure or dilutedin water for example) or in a mixture with other compounds (such asdietary supplements, nutritional supplements, medicines, carriers,flavors, digestible or non-digestible ingredients). Vitamins andminerals are examples of typical dietary supplements. In a preferredembodiment the composition is administered together with other compoundsthat enhance the described effect on the immunity of the offspring. Suchsynergistic compounds can be a carrier or a matrix that facilitates thedelivery of the probiotics to the intestinal tract of the female,preferably in an active form. Such synergistic compounds can also affectthe health status or metabolism of the expecting female such as toenhance the effect of the composition on the immune system of theoffspring. Such synergistic compounds can be other active compounds thatsynergistically or separately influence the immune response of theinfant and/or potentialize the effect of the probiotic. Example of suchsynergistic compounds is maltodextrine. One of the effects ofmaltodextrin is to provide a carrier for the probiotic, enhancing itseffect, and to prevent aggregation. Other examples include knownprebiotic compounds such as carbohydrate compounds selected from thegroup consisting of inulin, fructooligosaccharide (FOS), short-chainfructooligosaccharide (short chain FOS), galacto-oligosaccharide (GOS),xylooligosaccharide (XOS), glanglioside, partially hydrolysed guar gum(PHGG) acacia gum, soybean-gum, Lactowolfberry, wolfberry extracts ormixture thereof. Other carbohydrates can be present such as a secondcarbohydrate acting in synergy with the first carbohydrate and that isselected from the group consisting of xylooligosaccharide (XOS), gum,acacia gum, starch, partially hydrolysed guar gum or mixture thereof.

The carbohydrate or carbohydrates can be present at about 1 g to 20 g or1% to 80% or 20% to 60% in the daily doses of the composition.Alternatively the carbohydrates are present at 10% to 80% of the drycomposition. In any case however the daily dose of carbohydrate shouldcomply with the published safety guidelines and regulatory requirements.For children, a typical limit, for example, is a maximum of 6 g/L/day.

In an embodiment, a nutritional composition preferably comprises asource of protein. Dietary protein is preferred as a source of protein.The dietary protein may be any suitable dietary protein; for exampleanimal protein (such as milk protein, meat protein or egg protein);vegetable protein (such as soy protein, wheat protein, rice protein, andpea protein); a mixture of free amino acids; or a combination thereof.Milk proteins such as casein, whey proteins and soy proteins areparticularly preferred.

The composition may also comprise a source of carbohydrates and/or asource of fat.

If the composition of the invention is a nutritional composition andincludes a fat source, the fat source preferably provides about 5% toabout 55% of the energy of the nutritional composition; for exampleabout 20% to about 50% of the energy. Lipid making up the fat source maybe any suitable fat or fat mixture. Vegetable fat is particularlysuitable; for example soy oil, palm oil, coconut oil, safflower oil,sunflower oil, corn oil, canola oil, lecithin, and the like. Animal fatsuch as milk fat may also be added if desired.

An additional source of carbohydrate may be added to the nutritionalcomposition. It preferably provides about 40% to about 80% of the energyof the nutritional composition. Any suitable carbohydrate may be used,for example sucrose, lactose, glucose, fructose, corn syrup solids,maltodextrin or a mixture thereof.

Additional dietary fiber may also be added if desired. If added, itpreferably comprises up to about 5% of the energy of the nutritionalcomposition. The dietary fiber may be from any suitable origin,including for example soy, pea, oat, pectin, guar gum, acacia gum,fructooligosaccharide or a mixture thereof.

Suitable vitamins and minerals may be included in the nutritionalcomposition in an amount to meet the appropriate guidelines.

One or more food grade emulsifiers may be included in the nutritionalcomposition if desired; for example diacetyl tartaric acid esters ofmono- and di-glycerides, lecithin and mono- or di-glycerides or amixture thereof. Similarly suitable salts and/or stabilizers may beincluded. Flavors can be added to the composition.

Administration Period.

The administration period starts with the fecundation or as soon aspossible after fecundation (resp. after the mother-to-be is made awareof her pregnancy). However the administration period can also startearlier. For example, the administration period can precede thefecundation by 1 or 2 months. In such case, it is believed that thehealth status of the mother-to-be fecunded has an impact of the healthstatus of the offspring-to-be. The administration period can also startrelatively late in the pregnancy, preferably at month 3, 5 or 7 of thepregnancy (when considering human females) or in corresponding periodsfor other mammals. A very late start of the administration of thecomposition can also be considered, i.e. at or around month 8 or 9 (afew weeks before birth). In such case, it is speculated that the effecton the immune system of the offspring is a short term and rapid effect,best preparing it for the exposure to antigens after birth. The periodof administration can be continuous (for example up and includinglactation up to weaning), or discontinuous. Preferably the period ofadministration is continuous for better prolonged effect. However it isspeculated that a discontinuous pattern (for example dailyadministration during 1 week per month) can provide “discontinuousimmune boost signals” that induce positive effects on the offspring. Theduration of the administration may vary. If positive effects areexpected with relatively short duration of administration (for exampledaily administration during 1 week or 1 month), long durations arebelieved to provide enhanced effect (for example duration of 3, 5, or 8months in humans, and corresponding periods in other mammals).Preferably the administration period covers substantially the fulllength of the gestation period. In one embodiment it covers more than50%, more than 70%, or more than 80% of the gestation period. Theduration of administration can also cover all or a part of the lactationperiod.

Most preferably it also covers the full length of the lactation period.The duration can cover 0%, 30% or more, 50% or more or 80% or more ofthe lactation period. In one particular embodiment the period ofadministration of the composition can cover the lactation period (all orpart of it) but not the gestation period; in that case the benefits ofthe composition are transmitted through the female milk to theoffspring. Most preferably the administration period covers a part (orall) of the gestation period and part (or all) of the lactation period.Preferably the administration is be done in daily intake (to be takenonce or twice a day), or weekly intake (to be taken one or twice aweek).

In one embodiment, the composition is also administered directly to theoffspring provided that the mother has received the composition duringgestation. Preferably the composition is administered, most preferablyorally, directly to the offspring during lactation or after partial orfull weaning. The dual exposure of the mother (during gestation) and ofthe offspring (direct administration) to the composition can indeedprovide enhanced benefits in a synergistic way. It is speculated thatthe exposure during gestation induces a preconditioning of the offspringto better respond to the later direct administration.

Effect of the Composition.

The composition of the invention administered to expecting femalemammals induces the boost of the immunity of the offspring. That boostis particularly measurable after birth but may start during thegestation period.

Specifically, the term “boosting immunity” used therein excludes theallergic responses. The term “boosting immunity” is defined as boostingthe innate immune functions and promoting specific immune response toantigens. The innate immune response can be cellular responses,phagocytic activity, leucocytic activity and/or polyreactive antibodies.The specific immune response can be cell activation and/or specificantibody responses. The term “boosting immunity” can comprise or can bedefined as the enhancement of immune defenses of the body and/or theenhancement of the capability of the body to respond to infectiousantigenic challenges. The antigenic challenges can be viral and/orbacterial and/or parasitic agents or their antigen derivatives,sub-units, cell-surface compounds and/or toxins.

In one embodiment, the composition of the invention reinforces thetransmission of immune competences from the females to the offspring.Doing so, the composition is able to provide the offspring with betterchances to endure the challenges of their early life. Ultimately thecomposition helps the offspring to have a best start in life and/or tobetter protected against infections.

In one embodiment such boosting effect is an increase in the ability ofthe offspring to respond to an antigenic exposure. At birth the immunesystem of the infant is both in a state that enables him to respondappropriately to antigenic exposures and in a state of rapid maturation.The exposure to antigens also participates to enhance the maturation ofthe immune system. After birth all infants are naturally exposed toenvironmental or pathogenic antigens. The immune system of the offspringresponds to such exposure. In one embodiment of the present inventionthe immune system of the offspring is able to respond in a moreefficient way to exposure to antigens. In one embodiment the response tothe antigenic exposure can be measured by the dosage of antibodiesspecific to the antigen. In the context of the invention a quantitativeas well as qualitative elevation of the specific antibodies can bemeasured. In one embodiment such elevation can be measured in the serumand/or in the saliva and/or in the feces of the offspring exposed to theantigens. In one embodiment the response to the antigenic exposurecomprises an elevation of the total poly-reactive antibodies, preferablyin the serum and/or in the saliva and/or the feces of the offspring. Inone embodiment the response to the antigenic exposure comprises anelevation of the cellular immune response in the blood of the offspring,preferably an increase in number and/or increase in activity of theleucocytes of the offspring (for illustration, see results of example2). It is understood that the type of response is highly dependent onthe type of exposure, although some antigens can induce a complex immuneresponse measurable by more than one of the above cited effects (forexample elevation of specific antibodies and elevation of polyreactiveantibodies, and/or elevation of cellular immune response). Themeasurements of the immune response can be done by conventionally knownimmuno-assay methods, such as cellular counts, antibodies-assays,phagocytic activity as well as cytotoxic cell activity (neutrophils andnatural killer cells activity) dosage of immunity markers, includingcytokines, growth factors as well as cell-surface immune markers and thelike. The elevation is measured against the levels in the correspondingsamples from offspring which mothers were not exposed to the compositionof the invention (=control samples).

An antigenic exposure that is of particular relevance in the context ofthe invention includes but is not limited to exposure to virusespreferably rotaviruses and adenoviruses, or exposure to infectivebacteria, preferably Escherichia coli, Vibrio cholerae, salmonellae,clostridia, shigella, or exposure to infective parasites infection,preferably Giardia lamblia, Entamoeha histolytica and Cryptosporidiumspp or mixtures thereof. It is of particular interest for the healthstatus of the infants to exhibit a better ability to respond to suchpathogenic exposure. This can participate to a better protection of theoffspring against these pathogens, and ultimately to better protect themagainst the corresponding infections induced by these pathogens. Moregenerally it can participate to a better protection of the offspringagainst many types of pathogens (=enhanced immune system in general).Positive effect on allergies can also be contemplated within the scopeof this invention, as the effect on the immune system can be a betterbalanced and better controlled response to allergens.

The boosting effect on the immune system of the offspring can reach amaximum during the lactation period, or during juvenile phase/childhood.For humans the maximum of the boost is preferably achieved between birth(day 0) and months 24 of life, more preferably between birth (day 0) andday 180 of life. In is not excluded that the boosting effect can lastuntil the young adulthood phase of the life of the offspring. Fornon-human mammals corresponding periods have to be considered.

The invention will now be further illustrated by reference to thefollowing example:

Example 1

Mice Study 1

Material and Methods:

Conventional six-week-old female BALB/c mice (18-20 g) were used in allexperiments, purchased from Charles River (Domaine des Oncins, BP010969592. L'Arbresle Cedex, France). The mice were housed underspecific pathogen free conditions at the Clinique Medical Universitaireof Geneva (CMU-Geneva) animal facility, and 6 to 8 infant mice from eachlitter were allotted to each study group.

Animals had free access to regular conventional diet. Pregnant micereceived probiotic or placebo powders suspended in the drinking water.The drinking water was changed every day. Three groups were compared:

Group A control (Maltodextrine)

Group B Lactobacillus paracsei CNCM 1-2116 (ST11)

Group C Lactobacillus rhamnosus CGMCC 1.3724 (LPR)

All products were obtained from usual public source. The names “ST11”and “LPR” are abbreviations or nicknames for the cited microorganisms.

Live attenuated measles vaccine (MV-S) purchased from Aventis-Pasteur(Lyon, France).

Measurements

the determination of antibodies to measles virus (IgG1 and IgG2aisotypes) and of total serum IgG was performed by ELISA at the Centerfor vaccinology and Neonatal Immunology (CVNI) at University Medicalcenter of Geneva (Switzerland), according to standards.

Experimental Procedure:

(FIG. 1 provide a schematic view of the procedure):

1. Four pregnant mice per group received drinking water containing A, Bor C throughout pregnancy and during weaning.

2. At weaning (3 weeks) onward, all pups received regular water withoutany additives. Mothers were sacrificed.

3. At 3 weeks (infant immunization), pups (5-8 pups per litter; 28 pupsper group) were immunized with live attenuated measles virus (5×10⁵CCID₅₀).

4. Pups were monitored weekly (from 3 to 8 weeks of age) for weight gainand feces were collected once a week (from 3 to 8 weeks of age).

5. Pups were bled 3 and 5 weeks after immunization for determination oftotal IgG and of measles-specific IgG1 and IgG2a antibodies.

6. Five weeks after immunization all mice were sacrificed.

To evaluate differences measles vaccine, Krsukal-Wallis tests followedby Mann-Withney (Wilcoxon) tests were applied.

Results:

FIG. 2 provides a graphic illustration of the results obtained. Noeffect on the newborn's growth was identified. No differences in vaccineresponses related to litters, gender, or specific body weight wereobserved. A significant increase in antibody responses following measlesvaccination was observed in all groups, reflecting a normal antibodymaturation process. The immune response was characterized by a mixedTh1:Th2 responses as illustrated by the well-balanced IgG1 and IgG2alevels observed in all groups. Supplementation of mothers with ST11 hasno statistically significant effect on pup's responsiveness to measlesvaccine (under the testing conditions). However, LPR supplementation topregnant mothers promoted higher IgG titers compared to controls withoutaltering the general immune profile of the response to measles vaccine.

Conclusion:

These results seem to indicate that the effects are probiotic strainspecific. Indeed, ST11 seems to have little effect on pup's intestinalimmune maturation and responsiveness to measles vaccine. In contrast,LPR supplementation to pregnant mothers promoted increased peripheralIgG responses to measles vaccine in young adult mice.

Example 2

Mice Study 2

Material and Methods:

Conventional six-week-old pregnant female BALB/c mice (18-20 g) wereused in all experiments, purchased from Janvier, France. The mice werehoused under specific pathogen free conditions at the Nestle researchCenter animal facility, and 10 to 12 pups from each litter were allottedto each study group.

Animals had free access to regular conventional diet. Pregnant micereceived probiotic or placebo powders suspended in the drinking water.The drinking water was changed every day. Two groups were compared:

Group A control (Maltodextrine)

Group B Bifidobacterium lactis CNCM 1-3446 (BL)

All products were obtained from usual public source. The name “BL” is anabbreviation for the cited microorganism.

Tetanus Toxoid vaccine, Tetanol Pur (40 UI) was purchased from Novartis(Switzerland).

Measurements

The determination of spleen T-cell proliferation in presence of anti-CD3antibody was performed according to standard procedures. Briefly,splenocytes were incubated with plate-bound anti-mouse CD3 (2.5 g/mlincubated 2 h at 37° C., BD Pharmingen, cat no. 553056) in 96-wellplates at 37° C. for 72 h. Cell proliferation was determined by theincorporation of [methyl-3H]thymidine (Amersham Pharmacia Biosciences)as described (DeCicco, K. L., Youngdahl, J. D. and Ross, A. C. 2001Immunology 104, 341-348). Data are represented as counts per minute,normalized by the percentage of CD3-positive cells actually present inthe spleen of tested mice.

Dosages of serum IgG antibodies specific to Tetanus Toxoid wereperformed by ELISA. Briefly, 96-wells microtiter plates were coated with0.1 μg of whole TT antigen (Calbiochem, Tetanus Toxoid from Clostridiumtetani, cat no 582231) in PBS. After 3 washings with PBS-0.05% Tween20,plates were blocked with a PBS-20% FCS-0.05% Tween20 buffer for 1 h at37° C. and then washed 3 times again. Serial dilutions of mice sera inblocking buffer were incubated for 2 h at 37° C. After 3 washings, boundTT-specific IgG were detected by the incubation with a biotin-conjugatedgoat anti-mouse IgG antibody (SouthernBiotech, cat no. 1034-08) for 1 hat 37° C. Following 3 washings, ELISA plates were incubated withperoxidase labelled streptavidin (KPL, cat no. 14-30-00) for 30 minutesat 37° C. After 3 final washing steps, peroxidase substrate (KPL, catno. 50-76-00) was added. The colorimetric reaction was blocked withsulfuric acid and optical density at 450 nm was then measured in aspectro-photometer. Antibody titers were then calculated as described(Ma, Y. and Ross, A. C., 2005, Proc Natl Acad Sci. 2005 Sep. 20;102(38):13556-61).

Experimental Procedure:

(FIG. 3 provide a schematic view of the procedure):

1. Two to three pregnant mice per group received drinking watercontaining placebo or BL throughout pregnancy and during the first twoweeks of lactation.

2. At weaning (3 weeks) onward, all pups received regular water withoutany additives. Mothers were sacrificed.

3. At 3 weeks a sub-group of pups in each group (5-6 pups per group) wassacrificed to assess the spleen-cell proliferation.

4. At 3 weeks (infant immunization), remaining pups (5-6 pups per group)were immunized sub-cutaneously with TT vaccine (¼ of the human dose).

5. Four weeks after the first immunization a vaccine boost wasadministered to pups.

6. Pups were bled 4 weeks after the first immunization and 2 weeks afterthe boost for determination of anti-TT IgG antibodies, then the micewere sacrificed.

To evaluate differences in T-cell proliferation and IT vaccine aMann-Withney (Wilcoxon) tests were applied.

Results:

FIG. 4 show that a significant increase in antibody responses followingTT vaccination was observed in all groups, reflecting a normal antibodymaturation process. Supplementation of mums with BL during pregnancy andlactation promoted higher systemic T-cell reactivity and significantlyenhanced IgG titers compared to placebo.

Conclusions:

The two studies of examples 1 and 2 highlight the fact that it ispossible to promote immune development in offspring through perinatalintervention, in particular via supplementation of mothers duringpregnancy and lactation with probiotics. These effects arestrain-specific as indicated by the Study 1; however they seem not to bespecies-specific. Indeed. LPR or BL supplementation to pregnant mothersseems to promote a higher immune maturation in the offspring.

The invention is claimed as follows:
 1. A method for improving immunityin an offspring, the method comprising: administering to an expectingfemale mammal daily for all of the gestation period a compositioncomprising an effective amount of Lactobacillus rhamnosus CGMCC 1.3724so as to boost the immunity of the offspring after birth.
 2. The methodof claim 1 wherein the administering of the composition to the femalemammal induces boosting the innate immune functions and promotingspecific immune response to infectious antigens of the offspring.
 3. Themethod of claim 1 wherein the composition reinforces the transmission ofimmune competences from the female to the offspring.
 4. The method ofclaim 1 wherein the female mammal is human.
 5. The method of claim 1wherein the composition is administered orally to the female.
 6. Themethod of claim 1 wherein the composition is administered to the femalemammal for part of the lactation period of the offspring.
 7. The methodof claim 1 wherein the composition comprises ingredients selected fromthe group consisting of inulin, fructooligosaccharide (FOS), short-chainfructooligosaccharide (short chain FOS), galacto-oligosaccharide (GOS),xylooligosaccharide (XOS), glanglioside, partially hydrolysed guar gum,acacia gum, soybean-gum, Lactowolfberry, wolfberry extracts and mixturesthereof.
 8. The method of claim 1 wherein the immunity boost comprisesan increase in the ability of the offspring to respond to an antigenicexposure.
 9. The method of claim 8 wherein the response to the antigenicexposure comprises an elevation of the specific antibodies to theantigens in the serum, saliva or feces of the offspring.
 10. The methodof claim 8 wherein the response to the antigenic exposure comprises anelevation of the total poly-reactive antibodies in the offspring. 11.The method of claim 8 wherein the response to the antigenic exposurecomprises an elevation of cellular immune response in the blood of theoffspring.
 12. The method of claim 8 wherein the antigenic exposurecomprises exposure to at least one agent selected from the groupconsisting of viruses, bacteria, infective parasites infection andmixtures thereof.
 13. The method of claim 8 wherein the increasedability to respond to the antigenic exposure contributes to betterprotect the offspring against infections in early life.
 14. The methodof claim 1 wherein the immunity boost reaches its maximum during thelactation period, or during juvenile phase of the offspring.
 15. Themethod of claim 1 wherein the composition is further administered to theoffspring during a period selected from the group consisting oflactation, after a full weaning and after a partial weaning.
 16. Themethod of claim 1 wherein the composition is administered in a formselected from the group consisting of food, drinks, dietary supplementsand pharmaceutical compositions.
 17. The method of claim 1 wherein theimmunity boost reaches its maximum between birth (day 0) and day 180 oflife.
 18. A method for boosting the immunity of an offspring, the methodcomprising: administering to an expecting female mammal daily for all ofthe gestation period a composition comprising Lactobacillus rhamnosusCGMCC 1.3724 in an amount equivalent to between 10³ and 10¹⁰ cfu pergram of dry composition so as to boost the immunity of the offspringafter birth.